This invention concerns a method and apparatus for viewing phase phenomena. It is particularly suitable for use in viewing microscopic objects and the inventon will be described in detail in that context.
A phase phenomenon is one that alters the path length, but not the intensity, of a beam of time-varying radiation incident on it or passing through it as compared to a beam passing along the same path in the absence of such phenomenon. Although the invention conceivably could be practiced using sound waves, applications using a beam of electromagentic radiation are of far greater interest and the invention will be described in such terms in viewing microscopic objects. Any perfectly transparent object is a phase object since it alters the optical path length, but not the intensity, of a beam of electromagnetic radiation. Since the human eye detects variations in light intensity but is insensitive to phase variations, a purely phase object is invisible. Similarly, since photographic film is insensitive to phase variations, it is impossible to record the image of a phase object on film. In practice many objects which theoretically are phase objects will reduce the intensity of incident light by effects such as scattering, reflection and refraction making it possible to see these objects because of the resulting alterations in the intensity of light across the object. This, for example, is often how one discerns the presence of a plate glass window.
Phase phenomena may also be produced by reflecting a beam of incident radiation from an object having a uniformly reflective but contoured surface. Here, the surface contours alter the path length of the incident beam.
Numerous techniques are well known for the study of phase phenomena. In all these techniques radiation from the phase phenomenon being studied is modified so that phase differences caused by the object are converted to intensity differences in the image.
Most of these techniques are based on diffraction effects caused by the object being studied. As is well known, when a planar grating made of equi-spaced parallel straight opaque lines is illuminated by a collimated beam of light, some of that light is diffracted from its original path by the grating. The diffracted light travels along paths at equal angles to the right and left of the illuminating beam path in a plane perpendicular to the lines of the grating. The undiffracted light is known as the zeroeth order. The diffracted light to its right is the right sideband and that to the left is the left sideband. The various orders of diffracted light, first and higher, are in mirrored sequence in the two sidebands, symmetrically arrayed about the undiffracted beam.
Using a point source of light to illuminate a microscopic object, Ernst Abbe demonstrated that the light diffracted by the object carries the information specifying the shape of the object. The function of the microscope objective lens is to gather the diffracted light and the undiffracted part of the illuminating beam and redirect their paths so that they combine, without relative alteration in their optical path lengths, at the image plane. Here, interference between the undiffracted part of the illuminating beam and the diffracted light forms the image. Unless at least some of the light diffracted by the object is gathered by the objective, the object will not be resolved. He also observed, as exemplified in the case of extreme oblique illumination, that either sideband of the diffracted light can be combined with the undiffracted light to form the image even in the complete absence of the other sideband. Consideration of these facts led him to an understanding of the role of the numerical apertures of both the objective lens and the condenser lens in determining the resolving power of a microscope.
Subsequently, many noticed that phase objects were invisible under the conditions Abbe prescribed for maximum resolution. Zernicke examined this problem and found that the light diffracted by light absorbing objects had at the image plane a net phase displacement of 180 degrees relative to the undiffracted light. This produces destructive interference, thus accounting for the local intensity differences which make up the visible image. Zerincke reasoned that transparent objects, which do not produce visible intensity modulated images because they do not absorb light, must nevertheless produce images that recreate the phase displacements experienced by the light waves in passing through these objects. This could only occur if the light diffracted by these objects had at the image plane a net phase displacement of only 90 degrees. This conclusion led him to the development of the phase contrast microscope in which a phase plate located at the back focal plane of the objective produces an additional 90 degree phase shift between the diffracted light and the undiffracted part of the illuminating beam. This additional phase shift results in destructive interference and, therefore, a visible intensity modulated image.
Such a phase modification of the diffracted light can be accomplished in the rear focal plane of the objective lens of a microscope because the undiffracted beam is localized as an "image" of the light distribution in the front focal plane of the condenser lens. Thus, if the object is illuminated by directing a beam which propagates along the optical axis of the microscope through a circular aperture located in the front focal plane of a condenser lens, at the rear focal plane this beam will be localized as a spot of light at the center of the focal point, while the light diffracted by the object will be found throughout the focal point. The phase of the undiffracted beam can be modified relative to that of the diffracted light by inserting at the rear focal plane a phase plate which has a small central zone that is thicker or thinner than the surrounding region thereby increasing or decreasing the optical path length of the undiffracted light by the desired fraction of a wavelength. Similarly, if the object is illuminated by directing the illuminating beam through an annular aperture at the front focal plane of the condenser lens, as is presently preferred, the undiffracted beam may be modified at the rear focal plane by a phase plate having an annular zone of different optical path length. And, in general, to modify the phase of the undiffracted beam, the zone of different optical path length at the rear focal plane of the objective lens should match or image the aperture at the front focal plane of the condenser lens. Those skilled in the art will recognize the front focal plane of the condenser lens and the rear focal plane of the objective lens as Fourier transform planes. For further details on phase microscopy, see McGraw-Hill Encyclopedia of Science and Technology, Vol. 8, pp. 469-472 (3d edition), as well as Zernicke's chapter "The Wave Theory of Microscopic Image Formation" in J. Strong, Concepts of Classical Optics (Freeman, 1958).
Numerous modifications have been made to the basic phase contrast techniques. Although rectangular slit and cross shaped illuminating apertures and matching phase plates were initially used, contemporary commercial phase contrast microscopes use annular illuminating apertures and phase plates. Zernicke and others have suggested the use of attenuators in conjunction with the phase plates in the rear focal plane to modify the amplitude ratio between the diffracted beam and the undiffracted beam. See, for example, p. 533 of Zernicke's chapter cited above. Moreover, if the attenuator reduces the intensity of the undiffracted beam to nearly zero, dark field conditions will be achieved. However, since such attenuators are designed to produce only one amplitude ratio, they are inconvenient to use in situations where it is desirable to vary that ratio.
To alleviate this problem, others have used polarizers as variable attenuators in a structure in which a polarizer is placed before the condenser lens, an analyzer is located before the eyepiece and a polarizing filter is positioned in the rear focal plane of the objective. The filter comprises an annulus polarized in one direction with the center portion and periphery of the annulus polarized at right angles thereto. See, for example, A. H. Bennett, et al, Phase Microscopy, pp. 155-164 (Wiley 1951). The efficacy of such devices, however, is hampered by depolarization which takes place at the surface of each lens.
While phase contrast microscopes based on Zernicke's design still predominate as the most frequently used method for viewing microscopic phase objects, some of the peculiarities of the phase contrast images produced have prompted the development of other methods. Most frequently cited as objectionable is the halo of reversed contrast that borders image edges. Image duplicating interference microscopes have been developed to avoid this problem as well as to allow the quantitation of object optical thickness.
For example, in the interference microscope, interference is produced between two beams, one of which has been modified by a phase object. In the Linnik microscope for reflecting specimens, the Dyson microscope and the Smith-Baker microscope, one of these beams is a reference beam which does not illuminate the object while the other does. The two beams are combined by a semireflecting surface to cause interference which reveals surface contours in the case of reflecting specimens and differences in the optical path length through different portions of transparent specimens. Alternatively, both beams may illuminate the object at closely spaced but separate regions, in which case their interference reveals the rate of change of optical path length through different portions of transparent specimens as in the Nomarski microscope. The Linnik, Dyson and Smith-Baker microscopes are described in McGraw-Hill Encyclopedia of Science and Technology, Vol. 8, pp. 458-459 (3d edition). The Normarski microscope is described there and also in U.S. Pat. No. 2,924,142.
Because the angle at which the diffracted light diverges from the path of the illuminating beam increases directly as an arcsin function of the product of the wavelength of the illumination bean and the spatial frequency of the object detail responsible for diffraction, the portion of the diffracted light that can be gathered by an objective of given numerical aperture decreases as the spatial frequency of the object detail increases. Thus, the normal microscope objective with a full circular aperture and illuminated by a condenser of matching aperture acts as a spatial frequency filter favoring the low frequencies of the object structure, even though this combination extends the absolute limit of resolution (Abbe's limit) to the highest spatial frequencies possible for that objective. Consequently, while the image duplicating type of interference microscope eliminates the halo of the phase contrast microscope and has the desirable ability to quantitate optical path differences in the object, it does so at the expense of reduced visibility of the resolvable fine structure of the object. The differential interference contrast microscopes of Nomarski and Leitz-Smith preserve the use of the full objective and condenser aperture and produce high contrast for relatively fine specimen detail because its contrast generating mechanism constitutes a high pass spatial frequency filter. This is achieved at the cost of making the image resolution dependent on object orientation. On the whole, the differential interference microscope is capable of superior performance in imaging small phase objects but it requires exceptional care in construction and uses expensive crystalline components.
The modulation contrast system described by Hoffman is still another type of microscope for viewing phase objects. In this system light is directed through a rectangular slit at the condenser entrance pupil to illuminate the object; and then this light is attenuated by a three level attenuator located in the rear focal plane of the objective lens. The attenuator is circular in shape with a center band which permits limited transmission and two D-shaped regions on either side of the center band, one of which transmits incident light and the other of which blocks nearly all incident light. See, R. Hoffman et al., "The Modulation Contrast Microscope", Applied Optics, Vol. 14, No. 5 p. 1169 (May 1975).
Hoffman's theory for the operation of this device is that phase gradient objects in the specimen plane act as small prisms which refract the portion of the illuminating beam passing through them so that it falls either in the more transparent zone or in the more absorbing zone of the attenuator. In the image the regions, whose zero order light was refracted into the high transmission zones will appear brighter than the background, those whose zero order light was refracted into the low transmission zone will appear darker than the background and regions with constant optical thickness will not refract the zero order and will remain the same intensity as the background. This system has the virtue of simplicity but suffers from deficiencies in performance. This device is best suited for use at low magnifications for examining objects whose dimensions are much greater than the wavelength of light and whose image formation is therefore much less dependent on diffraction phenomena. At high magnifications it suffers from the limitations imposed by the use of a slit as entrance pupil. These are a susceptibility to disturbances in the image from objects out of the plane of focus and a strong dependence of resolution on specimen orientation relative to the slit.